1. Field of the Invention
This invention relates to the field of protein kinases and inhibitors thereof. In particular, the invention relates to inhibitors of phosphatidylinositol 3-kinase (PI3K) signaling pathways, and methods of their use.
2. Summary of the Related Art
The PI3K pathway regulates cell growth, proliferation and survival, and is dysregulated with high frequency in human tumors. PI3K pathway activation in tumors occurs via multiple mechanisms including prevalent mutation and amplification of the PIK3CA gene (which encodes the p110 subunit of PI3Ka), or downregulation of the lipid phosphatase PTEN. Downstream of PI3K, mTOR controls cell growth and proliferation through its two distinct signaling complexes: mTORC1 and mTORC2. Given the role of PI3K signaling on critical cellular functions, an inhibitor that targets both PI3K and mTOR could provide therapeutic benefit to patient populations with tumors harboring activating mutations in PIK3CA or Ras, PTEN-deletion, or where tumors are upregulated in growth factor signaling.
Phosphatidylinositol 3-kinase (PI3Kα), a dual specificity protein kinase, is composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. The protein encoded by this gene represents the catalytic subunit, which uses ATP to phosphorylate PtdIns, PtdIns4P and PtdIns(4,5)P2. PTEN, a tumor suppressor which inhibits cell growth through multiple mechanisms, can dephosphorylate PIP3, the major product of PIK3CA. PIP3, in turn, is required for translocation of protein kinase B (AKT1, PKB) to the cell membrane, where it is phosphorylated and activated by upstream kinases. The effect of PTEN on cell death is mediated through the PIK3CA/AKT1 pathway.
PI3Kα has been implicated in the control of cytoskeletal reorganization, apoptosis, vesicular trafficking, proliferation and differentiation processes. Increased copy number and expression of PIK3CA is associated with a number of malignancies such as ovarian cancer (Campbell et al., Cancer Res 2004, 64, 7678-7681; Levine et al., Clin Cancer Res 2005, 11, 2875-2878; Wang et al., Hum Mutat 2005, 25, 322; Lee et al., Gynecol Oncol 2005, 97, 26-34), cervical cancer, breast cancer (Bachman, et al. Cancer Biol Ther 2004, 3, 772-775; Levine, et al., supra; Li et al., Breast Cancer Res Treat 2006, 96, 91-95; Saal et al., Cancer Res 2005, 65, 2554-2559; Samuels and Velculescu, Cell Cycle 2004, 3, 1221-1224), colorectal cancer (Samuels, et al. Science 2004, 304, 554; Velho et al. Eur J Cancer 2005, 41, 1649-1654), endometrial cancer (Oda et al. Cancer Res. 2005, 65, 10669-10673), gastric carcinomas (Byun et al., Int J Cancer 2003, 104, 318-327; Li et al., supra; Velho et al., supra; Lee et al., Oncogene 2005, 24, 1477-1480), hepatocellular carcinoma (Lee et al., id.), small and non-small cell lung cancer (Tang et al., Lung Cancer 2006, 51, 181-191; Massion et al., Am J Respir Crit Care Med 2004, 170, 1088-1094), thyroid carcinoma (Wu et al., J Clin Endocrinol Metab 2005, 90, 4688-4693), acute myelogenous leukemia (AML) (Sujobert et al., Blood 1997, 106, 1063-1066), chronic myelogenous leukemia (CML) (Hickey and Cotter J Biol Chem 2006, 281, 2441-2450), and glioblastomas (Hartmann et al. Acta Neuropathol (Berl) 2005, 109, 639-642; Samuels et al., supra).
In view of the important role of PI3Kα in biological processes and disease states, inhibitors of this protein kinase are desirable.
The mammalian target of rapamycin, mTOR, is a protein kinase that integrates both extracellular and intracellular signals of cellular growth, proliferation, and survival. Extracellular mitogenic growth factor signaling from cell surface receptors and intracellular pathways that convey hypoxic stress, energy and nutrient status all converge at mTOR. mTOR exists in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is a key mediator of transcription and cell growth (via its substrates p70S6 kinase and 4E-BP1) and promotes cell survival via the serum and glucocorticoid-activated kinase SGK, whereas mTORC2 promotes activation of the pro-survival kinase AKT. Given its central role in cellular growth, proliferation and survival, it is perhaps not surprising that mTOR signaling is frequently dysregulated in cancer and other diseases (Bjornsti and Houghton Rev Cancer 2004, 4(5), 335-48; Houghton and Huang Microbiol Immunol 2004, 279, 339-59; Inoki, Corradetti et al. Nat Genet 2005, 37(1), 19-24).
mTOR is a member of the PIKK (PI3K-related Kinase) family of atypical kinases which includes ATM, ATR, and DNAPK, and its catalytic domain is homologous to that of PI3K. Dysregulation of PI3K signaling is a common function of tumor cells. In general, mTOR inhibition may be considered as a strategy in many of the tumor types in which PI3K signaling is implicated such as those discussed below.
In treating breast cancer, inhibition of PI3K signaling is critical for the activity of EGFR family inhibitors such as the anti-HER2 antibody trastuzumab (Nagata, Lan et al., Cancer Cell 2004, 6(2), 117-27), and loss of PTEN correlates with trastuzumab resistance (Pandolfi N Engl J Med 2004, 351(22), 2337-8; Nahta, Yu et al. Nat Clin Pract Oncol 2006, 3(5), 269-280). Therefore, inhibitors of PI3K signaling may be particularly useful in HER2 positive tumors that either fail to respond or become resistant to trastuzumab.
Mantle cell lymphoma (MCL) is usually characterized by hyperactivation of cyclin D and subsequent cell cycle dysregulation. Transcription of cyclin D is largely mediated by mTOR signaling, and rapamycin, an allosteric inhibitor of mTOR kinase activity, has been reported to downregulate cyclin D levels in MCL cell lines in vitro (Dal Col, Zancai et al. Blood 2008, 111(10), 5142-51).
In renal cell carcinoma, mTOR-promoted translation of hypoxia-inducible transcription factor (HIF1α), and resulting enhancement of the expression of vascular growth factors, may be particularly relevant in the case of tumors bearing loss of function mutations in the von Hippel-Lindau (VHL) protein. This protein serves to block proteaseome-mediated destruction of HIF and thereby causes constitutive expression of proangiogenic growth factors (Thomas, Tran et al. Nat Med 2006, 12(1), 122-7). Such mutations are particularly prevalent in renal cell carcinoma, and may underlie the promising clinical activity seen with mTOR inhibitors in this disease (Atkins, Hidalgo et al. J Clin Oncol 2004, 22(5), 909-18; Motzer, Hudes et al. J Clin Oncol 2007, 25(25), 3958-64).
PI3K is activated in blasts from acute myelogenous leukemia (AML) patients and may contribute not only to the pathophysiology of the disease but also to chemotherapy resistance. AML cells consistently express the p110δ isoform, and inhibition of PI3Kδ reduces proliferation and survival of AML cells without affecting normal hematopoietic progenitors (Sujobert, Bardet et al. Blood 2005, 106(3), 1063-6; Billottet, Grandage et al. Oncogene 2006, 25(50), 6648-6659). On the other hand, PI3K pathway activation has been associated with improved overall and relapse-free survival in newly diagnosed AML patients (Tamburini, Elie et al. Blood 2007, 110(3), 1025-8).
In chronic myelogenous leukemia (CML), the BCR-ABL oncogene signals through the p85 subunit of PI3K, mediating leukemogenesis, cell growth and cell survival (Skorski, Bellacosa et al. Embo J 1997, 16(20), 6151-61). A similar mechanism may be involved in growth and survival of NPM/ALK-transformed anaplastic large cell lymphoma cells (Bai, Ouyang et al. Blood 2000, 96(13), 4319-27). In addition, the p110γ subunit may be transcriptionally upregulated by BCR-ABL, and as such PI3Kγ, which is preferentially expressed in hematopoietic cells, may be an important target in drug-resistant CML (Hickey and Cotter Biol Chem 2006, 281(5), 2441-50).
The PI3K pathway was also found to be activated in diffuse large B cell lymphoma (DLBCL) cell lines and tumor samples, and PI3K inhibition led to apoptosis in several DLBCL cell lines (Uddin, Hussain et al. Blood 2006, 108(13), 4178-86).
Activation of the IGF1R receptor and downstream PI3K-mTOR pathway signaling is implicated in the genesis and progression of several subtypes of sarcoma (Hernando, Charytonowicz et al. Nat Med 2007, 13(6), 748-53; Wan and Helman Oncologist 2007, 12(8), 1007-18).
Rhabdomyosarcoma, one of the most common childhood sarcomas, is reported to be addicted to insulin-like growth factor I receptor signaling as measured by elevated AKT signaling (Cao, Yu et al. Cancer Res 2008, 68(19), 8039-8048). Insulin-like growth factor II is over-expressed in these cancers and the anti-tumor effect of rapamycin, an mTORC1 inhibitor, has been evaluated and reported to inhibit rhabdomyosarcoma xenograft growth (Wan, Shen et al. Neoplasia 2006, 8(5), 394-401).
Amplification of PIK3CA in ovarian cancer was one of the first indications that PI3Kα functions as a human oncogene (Shayesteh, Lu et al. Nat Genet, 1999, 21(1), 99-102). Both PI3K amplification and loss of PTEN have been associated with resistance to cisplatin in ovarian tumors (Lee, Choi et al. Gynecol Oncol 2005, 97(1) 26-34).
Both PTEN loss and PI3K mutation or amplification are common in endometrial tumors, with PTEN mutations found in up to 80% of the endometrioid subtype of endometrial carcinomas (Obata, Morland et al. Cancer Res 1998, 58(10), 2095-7). mTOR activation is quite common in endometrial tumors, promoted by downregulation of the LKB1 and/or TSC2 tumor suppressors, and dual inhibition of both the PI3K and mTOR axes may be a particularly useful strategy (Lu, Wu et al. Clin Cancer Res 2008, 14(9), 2543-50).
PI3K signaling may play an important role in many lung tumors; a recent study found overexpression of AKT and loss of PTEN in 41% and 46% of non small cell lung carcinoma (NSCLC) samples respectively (Tang, He et al. Lung Cancer 2006, 51(2), 181-91). Another recent analysis found 74% of NSCLC tumors with reduced or absent PTEN expression (Marsit, Zheng et al. Hum Pathol 2005, 36(7), 768-76). Frequent amplification of PIK3CA has also been demonstrated in various subtypes of lung cancer, including small cell (67%), squamous (70%), large cell (38%) and adenocarcinoma (19%) (Massion, Taflan et al. Am J Respir Crit Care Med 2004, 170(10), 1088-94). Notably, resistance to the EGFR inhibitor gefitinib correlates with failure to downregulate AKT signaling and loss of PTEN (Kokubo, Gemma et al. Br J Cancer 2005, 92(9), 1711-9), and KRAS status may also play an important role in determining response to EGFR inhibitors (Pao, Wang et al. Pub Library of Science Med 2005, 2(1), e17).
PI3K is mutated in 13-32% of colorectal tumors (Velho, Oliveira et al. Eur J Cancer 2005, 41(11), 1649-54; Foukas, Claret et al. Nature, 2006, 441(7091), 366-370), and loss of PTEN is observed in a high proportion of colorectal tumors, particularly those that display microsatellite instability (Goel, Arnold et al. Cancer Res 2004, 64(9), 3014-21; Nassif, Lobo et al. Oncogene 2004, 23(2), 617-28). Additionally, KRAS (which can also lead to upregulation of PI3K signaling) is the most frequently mutated oncogene in colorectal tumors (Bos Cancer Res 1989. 49(17), 4682-9; Fearon Ann N Y Acad Sci 1995, 768, 101-10). PI3K pathway dysregulation has also been implicated in impaired response to the anti-EGFR antibody cetuximab, and may also be of use in augmenting the response to chemotherapy (Perrone, Lampis et al. Ann Oncol 2009, 20(1), 84-90).
PTEN expression was found to be abnormally low in 36% of gastric carcinomas, and a similar proportion of tumors were found to have genomic amplifications of PIK3CA (Byun, Cho et al. Int J Cancer 2003, 104(3), 318-27).
PI3K activating mutations were reported to be found at high frequency in hepatocellular tumors (Lee, Soung et al. Oncogene 2005, 24(8), 1477-80). Furthermore, PTEN protein levels are decreased in up to 40% of liver tumors, and correlate inversely with pathological grade and disease progression (Hu, Huang et al. Cancer 2003, 97(8), 1929-40; Wan, Jiang et al. Cancer Res Clin Oncol 2003, 129(2), 100-6).
Loss of PTEN protein expression occurs in 18-19% of primary melanomas and 29-38% of melanoma cell lines, and is associated with increased tumor thickness (Guldberg, thor Straten et al. Cancer Res 1997, 57(17), 3660-3; Tsao, Zhang et al. Cancer Res 2000, 60(7), 1800-4; Whiteman, Zhou et al. Int J Cancer 2002, 99(1), 63-7; Goel, Lazar et al. J Invest Dermatol 126(1), 2006, 154-60).
Pancreatic tumors have a very high incidence of KRAS mutation, which elicits downstream activation of the PI3K pathway. In addition, decreased PTEN function has been reported in pancreatic cancer cell lines and tumor specimens, resulting in activated NFκB and stabilization of MYC (Asano, Yao et al. Oncogene 2004, 23(53), 8571-80).
Prostate carcinoma is characterized by frequent loss of PTEN function (Cairns, Okami et al. Cancer Res 1997, 57(22), 4997-5000; Gray, Stewart et al. Br J Cancer 1998, 78(10), 1296-300; Wang, Parsons et al. Clin Cancer Res 1998, 4(3), 811-5; Whang, Wu et al. Proc Natl Acad Sci USA 1998, 95(9), 5246-50), and PI3K inhibitors might have particular utility in this disease (Majumder and Sellers Oncogene 2005, 24(50) 7465-74). Recent data suggests that PTEN loss may play a fundamental role in expansion of a tumor stem cell-like population (Wang, Garcia et al. Proc Natl Acad Sci USA 2006, 103(5), 1480-5). There is extensive crosstalk between androgen receptor (AR) and PI3K pathway signaling, and PI3K/AKT or mTOR inhibition has shown promise in both androgen-dependent and independent preclinical models of prostate cancer (Lu, Ren et al. Int J Oncol 2006, 28(1), 245-51; Mulholland, Dedhar et al. Oncogene 25(3), 2006, 329-37; Xin, Teitell et al. Proc Natl Acad Sci USA 1 2006, 03(20), 7789-94; Mikhailova, Wang et al. Adv Exp Med Biol 2008, 617, 397-405; Wang, Mikhailova et al. Oncogene 2008, 27(56), 7106-7117).
PI3K is frequently mutated in thyroid carcinoma, particularly in the anaplastic subtype (Garcia-Rostan, Costa et al. Cancer Res 2005, 65(22), 10199-207). In a separate study, PI3K mutations were found to be relatively rare in thyroid arcinomas, but PI3K amplification was frequently found in follicular thyroid carcinoma (Wu, Mambo et al. J Clin Endocrinol Metab 2005, 90(8), 4688-93).
Anaplastic large cell lymphoma (ALCL) patients express an activated form of ALK kinase that results from a fusion of the nucleophosmin gene with the ALK kinase gene. The resulting fusion protine NPM-ALK is causally associated with ALCL and results in elevated mTOR signaling.
Hamaratomas, Angiomyelolipomas, TSC-Associated and Sporadic Lymphangioleiomyomatosis: Cowden's disease (multiple hamaratoma syndrome) is primarily the result of hereditary loss of function of PTEN. Patients develop hamaratomous neoplasms primarily of the skin and thyroid. Tuberous sclerosis patients develop benign hamaratomas in the lung, brain, kidneys, skin, and heart due to mutations in either TSC1 or TSC2. A large percentage of these patients also develop angiomyelolipomas (Bissler, McCormack et al. N Engl J Med 2008, 358(2), 140-151). Many female TSC patients also develop a progressive lung disease, lymphangioleiomyomatosis, which is a result of smooth muscle cell infiltration into the lungs (Bissler, et al. id.). While these tumors are benign, severe complications may arise as a consequence of the growth of these tumors and infiltrates.
Recent studies found that patients with sclerosing hemangioma, a rare lung tumor, have 84% reduced expression of STK11/LKB1 and elevated mTOR phosphorylation and signaling (Randa M. S. Amin Pathology International 2008, 58(1), 38-44). Germline mutations in LKB1/STK1 is the causal underlying mutation in Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder.
The primary form of treatment for head and neck cancer is surgery and/or radiation. There is evidence that radioresistance may occur via upregualtion of the PI3K/mTOR pathway (Gupta, McKenna et al. Clin Cancer Res 2002, 8(3), 885-892). Measurement of reduced AKT phosphorylation in patients correlated with better local control of the cancer.
In addition to the above tumor types, mTOR is particularly implicated in other diseases. For example, rapamycin is in clinical study for therapeutic treatment of neurofibromatosis. Mutation of the neurofibromatosis-1 gene in neurofibromatosis results in activation of the mTOR pathway (Ferner Eur J Hum Genet 2006, 15(2), 131-138; Sabatini Nat Rev Cancer 2006, 6(9), 729-734; Johannessen, Johnson et al. Current Biology 2008, 18(1), 56-62). In addition, rapamycin is in clinical study for macular degeneration, macular edema, and myeloid leukemia. Rapamycin is also being studied clinically in patients with systemic lupus and autoimmune lymphoproliferative syndrome (ALPS).